PROSPECTS OF LASSA FEVER CANDIDATE VACCINES

Background: Lassa fever is an acute viral haemorrhagic disease caused by the Lassa virus (LASV). It is endemic in West Africa and infects about 300,000 people each year, leading to approximately 5000 deaths annually. The development of the LASV vaccine has been listed as a priority by the World Health Organization since 2018. Considering the accelerated development and availability of vaccines against COVID-19, we set out to assess the prospects of LASV vaccines and the progress made so far. Materials and Methods: We reviewed the progress made on twenty-six vaccine candidates listed by Salami et al. (2019) and searched for new vaccine candidates through Google Scholar, PubMed, and DOAJ from June to July 2021. We searched the articles published in English using keywords that included “vaccine” AND “Lassa fever” OR “Lassa virus” in the title/abstract. Results: Thirty-four candidate vaccines were identified – 26 already listed in the review by Salami et al. and an additional 8, which were developed over the last seven years. 30 vaccines are still in the pre-clinical stage while 4 of them are currently undergoing clinical trials. The most promising candidates in 2019 were vesicular stomatitis virus-vectored vaccine and live-attenuated MV/LASV vaccine; both had progressed to clinical trials. Conclusions: Despite the focus on COVID-19 vaccines since 2020, LASV vaccine is under development and continues to make impressive progress, hence more emphasis should be put into exploring further clinical studies related to the most promising types of vaccines identified.


Introduction
Lassa fever (LF) is an acute viral hemorrhagic fever caused by the Lassa virus (LASV), a single-stranded RNA virus of the Arenaviridae family. The virus was first isolated in Nigeria in 1969 (World Health Organization, 2021). Rodents, particularly Mastomys natalensis, are the virus's natural hosts. The disease is primarily transmitted to humans through contact with infected rats' urine or feces (Ilori et al., 2019). Human-to-human infections and laboratory transmission can also occur, particularly in health care settings where infection prevention and control measures are insufficient (World Health Organization, 2021). LF is endemic in the West African countries of Ghana, Benin, Guinea, Liberia, Mali, Sierra Leone, and Nigeria (World Health Organization, 2021); an estimated 300,000 cases occur each year in this region, resulting in 5,000 deaths annually (Ilori et al., 2019). The number of sporadic cases occurring outside of the endemic regions within and outside of Africa is increasing due to an increase in international travel. In the United States, the United Kingdom (Geisbert et al., 2005) and Sweden (Asogun et al., 2019), these imported cases have been reported.
Lassa fever has an incubation period of 3-21 days. The early-stage disease is similar to other febrile diseases such as malaria and is usually only suspected after haemorrhagic symptoms develop in the late stages (Keïta et al., 2019). Fever, fatigue, haemorrhage, gastrointestinal symptoms (vomiting, diarrhoea, and stomach ache), respiratory symptoms (cough, chest pain and dyspnea), and neurologic symptoms (disorientation, seizures and unconsciousness) are all common clinical manifestations of the disease. However, cases of LF, especially in endemic areas, may remain asymptomatic (Ilori et al., 2019). In 2018, Nigeria had the highest number of cases of Lassa fever with 171 fatalities reported from the 633 confirmed cases (NCDC, 2018). Currently, there are no vaccines or effective drug therapies for the prevention or treatment of LF. Treatment with intravenous ribavirin has however been shown to reduce mortality and morbidity from LF if started within the first week of onset.
Lassa fever, one of the viruses causing severe haemorrhagic fever in Africa, is a major public health issue in endemic areas. When left untreated, LF can have a case fatality rate of up to 70% (Keïta et al., 2019), thus, a preventive vaccine is a critical public health need in endemic areas, particularly to protect health care providers, who are frequently the most at risk of exposure (Geisbert et al., 2005), Global efforts are being made to develop new vaccines for the control of diseases of global health importance (Salami et al., 2019). Lassa virus (LASV) vaccine development has been identified as a top priority by the World Health Organization (WHO) and the Coalition for Epidemic Preparedness Innovations (CEPI). A WHO Target Product Profile (TPP) of LASV vaccines was developed in June 2017 to aid the development of candidate vaccines for clinical evaluation. The TPP outlined the desired characteristics of potential LASV vaccines in two scenarios: preventive use and reactive/outbreak use. This led to an increase in LASV research, which has facilitated the development of vaccine candidates with pre-clinical proofs of concept that could potentially reduce illness, disease outbreaks, and deaths in humans (Salami et al., 2019).
According to the World Health Organization, LF vaccines should be cost-effective and affordable in endemic areas and stable for a reasonable time without extensive cold chain facilities. They should also provide immunity for special populations such as HIV-positive patients, pregnant women, and children. Their efficacy and protection must be at least 3 years; the method of administration should be simple, and a small number of doses should be required to confer long-term immunity (Salami et al., 2019). In 2019, Salami et al identified about 26 candidate vaccines for LF, some of which met the WHO criteria. Notably, the live-attenuated measles virus/Lassa virus (MV/LASV) platform was found to provide robust protection against LASV in animal models. Vesicular Stomatitis Virus (VSV)-vectored vaccine is another promising live virus vaccine platform, with at least two leading LASV vaccine candidates based on it (Salami et al., 2019). The most promising vaccine candidates were reported by (Hallam et al., 2018) to be live attenuated recombinant Mopeia virus/Lassa virus (MOPV/LASV) reassortment and live recombinant VSV and vaccinia vectored vaccines.
Most of the advanced vaccine candidates are expected to undergo human trials soon, though the clinical testing of these products and their deliveries to the most in-need population were reported to present significant problems (Salami et al., 2019). In this study, we will follow up on the 26 candidate vaccines identified by Salami and colleagues in 2019 to identify other potential candidate vaccines, discuss their prospects, their rates of development, and highlight factors affecting the development of Lassa fever vaccines.

Materials and Methods
We reviewed progress made on the 26 vaccine candidates listed by Salami and his colleagues in 2019 through Google Scholar, PubMed, and Directory of Open Access Journals (DOAJ) databases between June 23 rd , 2021 and July 7 th , 2021. We also searched for any new vaccine candidates using these databases between July 8 th , 2021 and July 17 th , 2021. We searched for articles published in English using keywords that included "vaccine" AND "Lassa Fever" OR "Lassa virus" in the title/abstract.

Lassa fever vaccines in development
In the review by Salami et al. (2019), 26 vaccines currently under development have been reviewed to check the present stage of development, target, and development partners. These are presented, along with progress made since then, in Table 1. This paper reviews the vaccine candidates and checks for progression into potential clinical trials.

rVSVN4CT1-LASV (VesiculoVaxTM Vesicular Stomatitis Virus Vector)
Vesicular Stomatitis Virus (VSV) is a virus infecting humans and animals and is an excellent candidate as a pseudovector. Five key proteins are encoded by the genomic RNA: phosphoprotein (P), glycoprotein (G), large protein (L), matrix protein (M), and nucleoprotein (N). Recombinant VSV equipped with a reporter gene instead of a VSV G gene in their genome makes it easier to evaluate infectivity in the study of viral entry, including the identification of viral receptors (Salami et al., 2019).
This vaccine has demonstrated usefulness in cynomolgus monkeys in an immune response against filoviruses including Ebola virus (EBOV), Sudan virus (SUDV), and Marburg virus (MARV). Further protection from LASV by a single vaccine is challenging because of several contributing factors. The virus is dissimilar to other species of filoviruses; that is why it has been classified as the Arenaviridae family. Overall, 4 groups of LASV have raised concerns about whether one vaccine will provide protection against the whole lineage (Cross et al., 2020).

ML29 L-AttV, rLCMV(IGR/S-S) (Mopiea/ Lassa reassortant)
ML29 is a live-attenuated vaccine (L-AttV) that was shown as a promising candidate in animal studies. Trials demonstrated that a single dose of the vaccine can provide sufficient protection against the virus. Nonetheless, as the ML29 attenuation mechanism is still not entirely understood, researchers have questioned if the vaccine has sufficient phenotypic stability to account for potential future mutations (Cai et al., 2020a).

VSV∆G/LASVGPC (VSV vector)
A phase I clinical trial to evaluate the safety and immunogenicity of rVSV∆G-LASV-GPC vaccine in adults in good general health commenced on June 23, 2021, sponsored by the International AIDS Vaccine Initiative (IAVI). IAVI's rVSV∆G-LASV-GPC uses a recombinant vesicular stomatitis virus (rVSV) vector just like the rVSVN4CT1-LASV vaccine. The same platform was used to produce Merck's highly efficacious Ebola Zaire virus vaccine, Ervebo®, recently approved by the European Commission, the United States Food and Drug Administration, regulators in several African countries, and prequalified by the World Health Organization (IAVI.ORG, n.d.).

YF 17D GPC
YF 17D GPC is based on the success of the Yellow Fever 17D (YF17D) vaccine in protecting humans against flaviviruses. The genetic skeleton for this vaccine candidate has been used for the construction of a bivalent YF17Dbased recombinant vaccine, which was engineered by combining LASV and YFV (Jiang et al., 2021). This vaccine, however, still does not show any immunogenicity in non-human primates. It is therefore necessary to explain and investigate the causes of low immunogenicity prior to considering it an option for the management of LSV in African regions (Purushotham et al., 2019).

ML29 virus
The reassortant ML29 carrying the L segment from the non-pathogenic Mopeia virus (MOPV) and the S segment from LASV is a vaccine candidate under development. ML29 demonstrated complete protection in validated animal models against a Nigerian strain from clade II, which was responsible for the most severe outbreak recorded in 2018 (Johnson et al., 2019).

LASV VLP
This virus-like particle vaccine is structurally and functionally similar to the LASV virus but lacks any replicative properties; hence, it can be safely recommended for use with low levels of biohazard risk. In a study by Branco et al. (2019) LASV VLP was immunogenic in mice in the absence of adjuvants, with mature IgG responses developing within a few weeks of the first immunization. These findings highlight the importance of using a VLP platform to develop the best vaccine candidates for Lassa hemorrhagic fever and they call for more research in lethal challenge animal models to establish their protective potential.

HLA-A02 and 10 HLA-A03-restricted epitopes
This vaccine is an attempt to develop one vaccine for the 7 strains of arenaviruses namely: Lymphocytic choriomeningitis virus (LCMV), Guanarito virus (GTOV), Junin virus (JUNV), Machupo virus (MACV), Sabia virus (SABV), Lassa and Whitewater Arroyo viruses. This can be achieved by choosing a molecule (epitope) common to them and targeting shared structural parts. HLA-transgenic mice were challenged following the peptide pool immunization and the magnitude of epitope-specific CD8+ T cell response was sufficient to reduce the viral titers. However, it was less extensive when contrasted with the responses found after individual peptide immunization (Salami et al., 2019).

LASSARAB
This vaccine has been designed to protect from both Lassa fever and rabies and was promising in the initial stages of testing. The candidate is based on a weakened, inactivated rabies vector. New studies indicate that LASSARAB injected with a GLA-SE adjuvant causes robust antibody production against both Lassa fever and rabies in guinea pigs and mouse experiments. Animals were protected from disease up to 58 days after administration (Abreu -Mota et al., 2018).
Previous research shows that immune responses are not usually proportionally correlated with protection from LSV. New studies, however, showed that high numbers of IgG antibodies which attach to the LSV proteins do in fact correlate with the protection from the virus. Thus, the levels of this antibody have the potential to be used to determine the usefulness and efficacy of the vaccine. The next step is to evaluate the experimental vaccine in nonhuman primates before advancing to human clinical trials (Abreu-Mota et al., 2018).

pLASV-GPC
DNA plasmid vaccination involves plasmid expressing gene(s) encoding the antigen(s) being introduced to cells, which stimulate an immune response (Cashman et al., 2017). pLASV-GPC encodes the GPC gene of the LF (Josiah strain), and when administered in 3 doses dermally, it protected guinea pigs from LF Virus-associated illness and death. The result was replicated in NHPs. Initially, this has been the most promising vaccine candidate as it is the first to commence human trials (Salami et al., 2019).

MV-LASV
The MV-LASV vaccine candidate is a recombinant live attenuated measles vectored vaccine, presenting antigen of the foreign pathogen which has progressed to clinical trials since Salami, et al's (2019) research. It offered strong protection in animal models demonstrating that viral replication and stimulation of innate immune responses are fundamental for an effective adaptive immune response in Lassa fever and has formed the basis for previously licensed vaccines (Henao-Restrepo et al., 2015). ChAdOx1 Lassa and ChAdOx1-biLAMA circumvent pre-existing immunity to human adenoviruses (Branco et al., 2010). Existing ChAdOx vaccines such as the ChAdOx1 nCoV-19 Covid-19 Vaccine which shows 70·4% efficacy against Covid-19 highlight the likely success of this blueprint for vaccines (Voysey et al., 2021).

Alphavirus replicon encoding LASV genes
The alphavirus replicon encoding Lassa Fever Virus genes is the only vaccine known to protect NHPs against at least three natural genotypes of Lassa virus (Lukashevich & Pushko, 2016).

Lassa GPCclamp
The Lassa GPCclamp-based vaccine has completed phase I clinical trials with 99% of vaccinated participants producing a neutralizing immune response. However, it has not progressed to phase II trials due to cross-reactivity in HIV patients.

LHF-535
A small-molecule viral entry inhibitor was developed to protect against the Lassa virus by targeting the viral envelope glycoprotein. It is currently the only viral entry inhibitor vaccine for the Lassa virus. Since its development, the phase I clinical trial has been completed and reported to be well tolerated (Health Newswire, 2021; Madu et al., 2018).

MVALassaNP
The MVALassaNP was found to trigger both humoral and cell-mediated immunity against the Lassa virus. It was proposed to be protective against the Lassa virus due to its cell-mediated function and also halted disease progression in pigs (Kennedy et al., 2019).

Lassa VRPs
The vaccine combines the benefits of a live vaccine with that of a single cycle of replication. It demonstrated protective immunity against the disease in lethal guinea pig models (Kainulainen et al., 2018).

MeV-NP
MeV-NP was developed by generating MeV expressing NP protein. It induced efficient protection in a single dose in monkeys (Mateo et al., 2019).

rLASV-GPC/CD LAV
Researchers from the University of Rochester and Schipps Research Institute adopted a model used to develop a recombinant form of the mammarenavirus lymphocytic choriomeningitis virus (rLCMV) using a colon deoptimization glycoprotein (CD GPC ). They incorporated the CD-GPC to develop rLASV-GPC/CD (Cai et al., 2020b). In low single doses, the vaccine offered complete protection to Hartley guinea pigs (Cai et al., 2020a). Currently, the vaccine is funded by the Coalition for Epidemic Preparedness Innovations (CEPI) and is still in the preclinical phase.

ChAdOx1-Lassa-GPC
This vaccine comprises of proprietary chimpanzee adenovirus vector platform (ChAdOx1) expressing the Josiah strain LASV GPC: ChAdOX1-Lassa-GP, this vector is derived from chimpanzee adenovirus Y25 (Purushotham et al., 2019). ChAdOx1-vectored Lassa fever vaccine encodes the full-length Josiah strain LASV GPC sequence (ChAdOx1-Lassa-GPC) and protective efficacy was seen after one single dose was given to guinea pigs (Fischer et al., 2021). The vaccine was developed by the University of Oxford and Janssen Vaccines & Prevention B.V. and is being funded by CEPI (Purushotham et al., 2019).

IN0-4500(DNA)
IN0-4500 was developed by Inovio's Pharmaceutical and encodes the LASV Josiah strain, GPC gene, funded by CEPI. It was demonstrated to offer 100% efficacy after two immunizations at four sites (Purushotham et al., 2019). It is still in the preclinical phase of development (Bernasconi et al., 2020).

Discussion
There is substantial progress in the development of LF vaccines using a variety of novel technologies. In the review by Salami et al. (2019), twenty-six vaccine candidates were identified in preclinical stages. Since then, three of them have progressed to clinical trials, with one securing funding for Phase 2 trials. Most vaccine candidates have significantly progressed since 2019 with only a few being withdrawn. Out of eight new candidates included in Table 2., one vaccine candidate has progressed to clinical Phase 1 trial.
The most promising candidates in 2019 were Vesicular Stomatitis Virus (VSV)-vectored vaccine and Liveattenuated MV/LASV have already progressed to clinical trials. Other candidates that have seen progress include Mopeia, Lassa Virus reassortants with various antigens as well as DNA platforms. The review of new candidates has shown that additional eight vaccines have been designed, seven of them currently in preclinical stages. Some of these are already being evaluated for clinical phases due to successful trials on animal subjects. We, therefore, recommend that further studies be tailored around the four types which have successfully progressed to clinical trials.

Conclusions
To be used in endemic regions, the vaccine must be cost-effective, affordable, and sustainable according to the WHO's target product profiles (World Health Organization, 2019). The vaccine development has been described as crucial by the WHO since LF was identified as a potential risk for a pandemic. Following the problems in vaccine production, particularly that posed by LSV lineage diversity, there has been an increased demand to see clinical candidates for vaccines that can safely target multiple strains of the virus. It is critical that all vaccines based on the LASV Josiah strain are tested across all lineages to ensure universality. To conclude, since 2019 and despite the focus on the development of COVID-19 vaccines globally, some of the Lassa virus vaccine candidates have significantly progressed in both preclinical and clinical tests shaping a profoundly mature pipeline in terms of safety and usefulness.